E.C. Zwarthoff (Ellen)http://repub.eur.nl/ppl/921/
List of Publicationsenhttp://repub.eur.nl/eur_signature.pnghttp://repub.eur.nl/
RePub, Erasmus University RepositoryTERT promoter mutations and BRAF mutations are rare in sporadic, and TERT promoter mutations are absent in NF1-related malignant peripheral nerve sheath tumorshttp://repub.eur.nl/pub/70081/
Fri, 18 Jul 2014 00:00:01 GMT<div>H.J. Dubbink</div><div>H. Bakels</div><div>E. Post</div><div>E.C. Zwarthoff</div><div>R.M. Verdijk</div>
Hot spot mutations in the promoter region of telomerase reverse transcriptase (TERT promoter mutations) occur frequently in tumors of neuroectodermal origin such as melanoma and glioma. Many of these tumors are of neuroectodermal or ectomesenchymal origin which is suggestive of TERT promoter mutations playing a role in the development of malignant peripheral nerve sheath tumors (MPNSTs). In melanoma a correlation has been suggested between the occurrence of TERT promoter mutations and v-RAF murine sarcoma viral oncogene homolog B1 (BRAF) mutations. We investigated TERT promoter and BRAF mutation frequency in respectively 94 and 86 consecutive MPNST cases from our institute. TERT promoter mutation analysis on DNA from formalin-fixed, paraffin-embedded specimens was performed by SNaPshot analysis. Sequence analysis of BRAF was performed by bidirectional DNA sequencing. We identified TERT C228T or C250T promoter mutations in 10 % (9/94) and BRAF V600E mutations in 3 % (3/86) of MPNSTs. All TERT promoter- and BRAF mutations occurred in NF1 unrelated tumors. One co-occurrence of a TERT promoter- and a BRAF mutation was observed. In comparison with other neuroectodermal derived malignant neoplasms, TERT promoter mutations occur at relatively low frequency in MPNSTs. The observation of TERT promotor and BRAF mutations in sporadic MPNSTs and the absence of TERT promotor and rarity of BRAF mutations in NF1 related tumors may imply an alternative genetic route of tumor progression in both patient groups.Risk prediction scores for recurrence and progression of non-muscle invasive bladder cancerhttp://repub.eur.nl/pub/56506/
Fri, 06 Jun 2014 00:00:01 GMT<div>M.M. Vedder</div><div>M. Márquez</div><div>E.W. de Bekker-Grob</div><div>M.L. Calle</div><div>L. Dyrskjot</div><div>M. Kogevinas</div><div>U. Segersten</div><div>P.-U. Malmström</div><div>F. Algaba</div><div>W. Beukers</div><div>T.F. Orntoft</div><div>E.C. Zwarthoff</div><div>F.X. Real</div><div>N. Malats</div><div>E.W. Steyerberg</div>
Objective: We aimed to determine the validity of two risk scores for patients with non-muscle invasive bladder cancer in different European settings, in patients with primary tumours. Methods: We included 1,892 patients with primary stage Ta or T1 non-muscle invasive bladder cancer who underwent a transurethral resection in Spain (n = 973), the Netherlands (n = 639), or Denmark (n = 280). We evaluated recurrence-free survival and progression-free survival according to the European Organisation for Research and Treatment of Cancer (EORTC) and the Spanish Urological Club for Oncological Treatment (CUETO) risk scores for each patient and used the concordance index (c-index) to indicate discriminative ability. Results: The 3 cohorts were comparable according to age and sex, but patients from Denmark had a larger proportion of patients with the high stage and grade at diagnosis (p<0.01). At least one recurrence occurred in 839 (44%) patients and 258 (14%) patients had a progression during a median follow-up of 74 months. Patients from Denmark had the highest 10-year recurrence and progression rates (75% and 24%, respectively), whereas patients from Spain had the lowest rates (34% and 10%, respectively). The EORTC and CUETO risk scores both predicted progression better than recurrence with c-indices ranging from 0.72 to 0.82 while for recurrence, those ranged from 0.55 to 0.61. Conclusion: The EORTC and CUETO risk scores can reasonably predict progression, while prediction of recurrence is more difficult. New prognostic markers are needed to better predict recurrence of tumours in primary non-muscle invasive bladder cancer patients.Absence of TERT promoter mutations in esophageal adenocarcinomahttp://repub.eur.nl/pub/52550/
Tue, 15 Apr 2014 00:00:01 GMT<div>A.M.J. van Nistelrooij</div><div>E.C. Zwarthoff</div><div>E. Post</div><div>I. Lurkin</div><div>R. van Marion</div><div>E. Kopershoek</div><div>K. Biermann-Pauls</div><div>B.P.L. Wijnhoven</div><div>W.N.M. Dinjens</div>
Telomerase reverse transcriptase promoter mutations in bladder cancer: High frequency across stages, detection in urine, and lack of association with outcomehttp://repub.eur.nl/pub/73144/
Sat, 01 Feb 2014 00:00:01 GMT<div>Y. Allory</div><div>W. Beukers</div><div>A. Sagrera</div><div>M. Flández</div><div>M. Marqués</div><div>M. Márquez</div><div>K.A. van der Keur</div><div>L. Dyrskjot</div><div>I. Lurkin</div><div>M. Vermeij</div><div>A. Carrato</div><div>J. Lloreta</div><div>J.A. Lorente</div><div>E. Carrillo-de Santa Pau</div><div>R.G. Masius</div><div>M. Kogevinas</div><div>E.W. Steyerberg</div><div>A.A. van Tilborg</div><div>C.S. Abas</div><div>T.F. Orntoft</div><div>T.C.M. Zuiverloon</div><div>N. Malats</div><div>E.C. Zwarthoff</div><div>F.X. Real</div>
Background Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression. Objectives To investigate TERT mutation frequency, spectrum, association with expression and clinical outcome, and potential for detection of recurrences in urine in patients with urothelial bladder cancer (UBC). Design, setting, and participants A set of 111 UBCs of different stages was used to assess TERT promoter mutations by Sanger sequencing and TERT messenger RNA (mRNA) expression by reverse transcription-quantitative polymerase chain reaction. The two most frequent mutations were investigated, using a SNaPshot assay, in an independent set of 184 non-muscle-invasive and 173 muscle-invasive UBC (median follow-up: 53 mo and 21 mo, respectively). Voided urine from patients with suspicion of incident UBC (n = 174), or under surveillance after diagnosis of non-muscle-invasive UBC (n = 194), was tested using a SNaPshot assay. Outcome measurements and statistical analysis Association of mutation status with age, sex, tobacco, stage, grade, fibroblast growth factor receptor 3 (FGFR3) mutation, progression-free survival, disease-specific survival, and overall survival. Results and limitations In the two series, 78 of 111 (70%) and 283 of 357 (79%) tumors harbored TERT mutations, C228T being the most frequent substitution (83% for both series). TERT mutations were not associated with clinical or pathologic parameters, but were more frequent among FGFR3 mutant tumors (p = 0.0002). There was no association between TERT mutations and mRNA expression (p = 0.3). Mutations were not associated with clinical outcome. In urine, TERT mutations had 90% specificity in subjects with hematuria but no bladder tumor, and 73% in recurrence-free UBC patients. The sensitivity was 62% in incident and 42% in recurrent UBC. A limitation of the study is its retrospective nature. Conclusions Somatic TERT promoter mutations are an early, highly prevalent genetic event in UBC and are not associated with TERT mRNA levels or disease outcomes. A SNaPshot assay in urine may help to detect UBC recurrences.Defined morphological criteria allow reliable diagnosis of colorectal serrated polyps and predict polyp geneticshttp://repub.eur.nl/pub/71922/
Wed, 01 Jan 2014 00:00:01 GMT<div>T.T. Rau</div><div>A. Agaimy</div><div>A. Gehoff</div><div>C. Geppert</div><div>K. Jung</div><div>K. Knobloch</div><div>C. Langner</div><div>A. Lugli</div><div>I. Groenbus-Lurkin</div><div>I.D. Nagtegaal</div><div>J. Rüschoff</div><div>X. Saegert</div><div>M. Sarbia</div><div>R. Schneider-Stock</div><div>M. Vieth</div><div>E.C. Zwarthoff</div><div>A. Hartmann</div>
Criteria for the diagnosis of serrated colorectal lesions (hyperplastic polyp, sessile serrated adenoma without or with dysplasia-which we called mixed polyp-and traditional serrated adenoma) for which consensus has been reached should be validated for applicability in daily practice in terms of inter-observer reproducibility and their association with clinical features and (epi)genetic events. A study set was created from a consecutive series of colorectal polyps (n=1,926) by selecting all sessile serrated adenomas, traditional serrated adenomas and mixed polyps. We added consecutive series of hyperplastic polyps, classical adenomas and normal mucosa samples for a total of 200 specimens. With this series, we conducted an inter-observer study, encompassing ten pathologists with gastrointestinal pathology experience from five European countries, in three rounds in which all cases were microscopically evaluated. An assessment of single morphological criteria was included, and these were correlated with clinical parameters and the mutation status of KRAS, BRAF and PIK3CA and the methylation status of MLH1. Gender, age and localisation were significantly associated with certain types of lesions. Kappa statistics revealed moderate to good inter-observer agreement for polyp classification (κ = 0.56 to 0.63), but for single criteria, this varied considerably (κ = 0.06 to 0.82). BRAF mutations were frequently found in hyperplastic polyps (86 %, 62/72) and sessile serrated adenomas (80 %, 41/51). KRAS mutations occurred more frequently in traditional serrated adenomas (78 %, 7/9) and less so in classical adenomas (20 %, 10/51). Single morphological criteria for sessile serrated adenomas showed significant correlation with BRAF mutation (all p≤0.001), and those for classical adenomas or traditional serrated adenoma correlated significantly with KRAS mutation (all p<0.001). Therefore, single well-defined morphological criteria are predictive for genetic alterations in colorectal polyps.HRAS mutations in bladder cancer at an early age and the possible association with the Costello Syndromehttp://repub.eur.nl/pub/56278/
Wed, 30 Oct 2013 00:00:01 GMT<div>W. Beukers</div><div>A. Hercegovac</div><div>E.C. Zwarthoff</div>
The Use of Molecular Analyses in Voided Urine for the Assessment of Patients with Hematuriahttp://repub.eur.nl/pub/74067/
Tue, 15 Oct 2013 00:00:01 GMT<div>W. Beukers</div><div>R. Kandimalla</div><div>D. van Houwelingen</div><div>H. Kovacic</div><div>J.-F.D. Chin</div><div>H.F. Lingsma</div><div>L. Dyrskjot</div><div>E.C. Zwarthoff</div>
Introduction:Patients presenting with painless hematuria form a large part of the urological patient population. In many cases, especially in younger patients, the cause of hematuria is harmless. Nonetheless, hematuria could be a symptom of malignant disease and hence most patients will be subject to cystoscopy. In this study, we aimed to develop a prediction model based on methylation markers in combination with clinical variables, in order to stratify patients with high risk for bladder cancer.Material and Methods:Patients (n=169) presenting with painless hematuria were included. 54 patients were diagnosed with bladder cancer. In the remaining 115 patients, the cause of hematuria was non-malignant. Urine samples were collected prior to cystoscopy. Urine DNA was analyzed for methylation of OSR1, SIM2, OTX1, MEIS1 and ONECUT2. Methylation percentages were calculated and were combined with clinical variables into a logistic regression model.Results:Logistic regression analysis based on the five methylation markers, age, gender and type of hematuria resulted in an area under the curve (AUC) of 0.88 and an optimism corrected AUC of 0.84 after internal validation by bootstrapping. Using a cut-off value of 0.307 allowed stratification of patients in a low-risk and high-risk group, resulting in a sensitivity of 82% (44/54) and a specificity of 82% (94/115). Most aggressive tumors were found in patients in the high-risk group. The addition of cytology to the prediction model, improved the AUC from 0.88 to 0.89, with a sensitivity and specificity of 85% (39/46) and 87% (80/92), retrospectively.Conclusions:This newly developed prediction model could be a helpful tool in risk stratification of patients presenting with painless hematuria. Accurate risk prediction might result in less extensive examination of low risk patients and thereby, reducing patient burden and costs. Further validation in a large prospective patient cohort is necessary to prove the true clinical value of this model.A 3-plex methylation assay combined with the FGFR3 mutation assay sensitively detects recurrent bladder cancer in voided urinehttp://repub.eur.nl/pub/41434/
Sun, 01 Sep 2013 00:00:01 GMT<div>R. Kandimalla</div><div>R.G. Masius</div><div>W. Beukers</div><div>C.H. Bangma</div><div>T.F. Orntoft</div><div>L. Dyrskjot</div><div>N. van Leeuwen</div><div>B. Roozenbeek</div><div>A.A. van Tilborg</div><div>E.C. Zwarthoff</div>
Purpose: DNA methylation is associated with bladder cancer and these modifications could serve as useful biomarkers. FGFR3 mutations are present in 60% to 70% of non-muscle invasive bladder cancer (NMIBC). Low-grade bladder cancer recurs in more than 50% of patients. The aim of this study is to determine the sensitivity and specificity of a urine assay for the diagnosis of recurrences in patients with a previous primary NMIBC G1/G2 by using cystoscopy as the reference standard. Experimental Design: We selected eight CpG islands (CGI) methylated in bladder cancer from our earlier genome-wide study. Sensitivity of the CGIs for recurrences detection was investigated on a test set of 101 preTUR urines. Specificity was determined on 70 urines from healthy males aged more than 50 years. A 3-plex assay for the best combination was developed and validated on an independent set of 95 preTUR, recurrence free, and nonmalignant urines (n = 130). Results: The 3-plex assay identified recurrent bladder cancer in voided urine with a sensitivity of 74% in the validation set. In combination with the FGFR3 mutation assay, a sensitivity of 79% was reached (specificity of 77%). Sensitivity of FGFR3 and cytology was 52% and 57%, respectively. Conclusion: The combination of methylation and FGFR3 assays efficiently detects recurrent bladder cancer without the need for stratification of patients regarding methylation/mutation status of the primary tumor. We conclude that the sensitivity of this combination is in the same range as cystoscopy and paves the way for a subsequent study that investigates a modified surveillance protocol consisting of the urine test followed by cystoscopy only when the urine test is positive.Outcomes of a bladder cancer screening program using home hematuria testing and molecular markershttp://repub.eur.nl/pub/40956/
Mon, 01 Jul 2013 00:00:01 GMT<div>C.H. Bangma</div><div>S. Loeb</div><div>M. Busstra</div><div>X.D. Zhu</div><div>S. el Bouazzaoui</div><div>J. Refos</div><div>K.A. van der Keur</div><div>S.S. Tjin</div><div>C.G.A.M. Franken</div><div>G.J.H.L. Leenders</div><div>E.C. Zwarthoff</div><div>M.J. Roobol-Bouts</div>
Background: We previously reported the preliminary findings from a feasibility study of bladder cancer (BCa) screening with urinary molecular markers (Bladder Cancer Urine Marker Project [BLU-P]) that has now been terminated. Objective: To report the final results from BLU-P to determine whether mass screening for BCa is feasible and useful. Design, setting, and participants: BLU-P was a Dutch population-based study initiated in 2008 to evaluate BCa screening. A total of 6500 men were invited to participate in the study, 1984 (30.5%) agreed, and 1747 (88.1%) men completed the protocol and were followed for 2 yr. Intervention: The screening protocol included home hematuria testing followed by molecular markers - nuclear matrix protein 22 (NMP22), microsatellite analysis (MA), fibroblast growth factor receptor 3 (FGFR3) mutation snapshot assay, and a custom methylation-specific (MLPA) test - to determine the need for cystoscopy. Outcome measurements and statistical analysis: Outcomes included the number of cystoscopies and the cancer detection rate within and outside the protocol, as determined by linkage to national registries. Results and limitations: Overall, 409 men (23.4%) tested positive for hematuria and underwent molecular testing. Current smokers (n = 295 [17%]) and past smokers (n = 998 [58%]) were significantly more likely to test positive for hematuria than nonsmokers. Seventy-one of 75 men (94.6%) with positive molecular markers underwent the recommended cystoscopy. Four BCas and one kidney tumor were detected through this sequential protocol, whereas one BCa and one kidney tumor were missed through the screening program. Limitations include the possibility of healthy subject bias. Conclusions: For BCa screening, use of a sequential protocol with home hematuria testing followed by molecular markers substantially reduced the number of cystoscopy recommendations compared with dipstick testing alone. A sequential screening approach may help minimize unnecessary invasive follow-up testing, with very few missed cancers. Nevertheless, this mass screening program had a very low diagnostic yield in an unselected asymptomatic European male population. Hypermethylation of the polycomb group target gene PCDH7 in bladder tumors from patients of all ageshttp://repub.eur.nl/pub/73723/
Mon, 01 Jul 2013 00:00:01 GMT<div>W. Beukers</div><div>A. Hercegovac</div><div>M. Vermeij</div><div>R. Kandimalla</div><div>A.C. Blok</div><div>M.M.N. van der Aa</div><div>E.C. Zwarthoff</div><div>T.C.M. Zuiverloon</div>
Purpose: Bladder tumors in patients younger than 20 years show a low incidence of the genetic and epigenetic aberrations typically found in older patients. One of the most common epigenetic aberrations in human malignancies is DNA hypermethylation. Polycomb group complexes have an important role during lineage choices in embryogenesis and their target genes are 12 times more likely to be methylated than nonpolycomb group target genes. We hypothesized that methylation of polycomb group target genes is an early event in urothelial carcinogenesis and thus might be observed in young patients. Materials and Methods: We stratified 167 patients by age into 4 groups, including age less than 20 years in 14, 20 to 40 in 48, 40 to 60 in 47 and greater than 60 in 58. Five previously identified polycomb group target genes (MEIS1, ONECUT2, OTX1, PCDH7 and SOX21) were selected for methylation analysis. Methylation ratios were calculated by using the unmethylated and methylated signal. The outcome represented the fraction of methylated cells within one tumor. Genes with similar methylation ratios in all age groups were considered as potential bladder cancer initiating candidates. Results: Three genes showed higher methylation ratios in tumors from older patients, including ONECUT2, SOX21 and OTX1 (each p <0.001). MEIS1 showed a similar methylation ratio in all groups but the median methylation ratio was low. PCDH7 showed a similar median methylation percent in all age categories, ie 54% at less than 20, 59% at 20 to 40, 59% at 40 to 60 and 67% at greater than 60 years (p = 0.1). Conclusions: Tumors from young patients showed less methylation for most markers. PCDH7 showed high methylation ratios in all age categories. Therefore, it could have an important role in early urothelial carcinogenesis.Combinations of urinary biomarkers for surveillance of patients with incident nonmuscle invasive bladder cancer: The European FP7 UROMOL projecthttp://repub.eur.nl/pub/62311/
Wed, 01 May 2013 00:00:01 GMT<div>T.C.M. Zuiverloon</div><div>W. Beukers</div><div>K.A. van der Keur</div><div>A.J.M. Nieuweboer</div><div>T. Reinert</div><div>L. Dyrskjot</div><div>T.F. Orntoft</div><div>E.C. Zwarthoff</div>
Purpose: We determined a combination of markers with optimal sensitivity to detect recurrence in voided urine after resection of an incident low grade, nonmuscle invasive bladder tumor. Materials and Methods: A total of 136 patients with G1/G2 nonmuscle invasive bladder tumor were included in the study at transurethral resection of the incident tumor. At least 3 followup urine samples were required for patient selection. DNA was extracted from the incident tumor and cell pellets of subsequently collected urine samples. We performed FGFR3, PIK3CA and RAS mutation analysis, and microsatellite and methylation analysis on tissue and urine DNA samples. Results: We obtained 716 urine samples. The 136 patients experienced a total of 552 recurrences during a median 3-year followup. Sensitivity for detecting a recurrent tumor varied between 66% and 68% for the molecular tests after patient stratification based on tumor DNA analysis. A combination of markers increased sensitivity but decreased the number of patients eligible for a certain test combination. Combining urine cytology with FGFR3 analysis without stratifying for FGFR3 status of the incident tumor increased sensitivity from 56% to 76%. Conclusions: A combination of markers increased the percentage of patients eligible for urine based followup and the sensitivity of recurrence detection. Adding FGFR3 analysis to urine cytology could be valuable for noninvasive followup of patients with nonmuscle invasive bladder cancer.DNA methylation-based biomarkers in bladder cancerhttp://repub.eur.nl/pub/40156/
Tue, 30 Apr 2013 00:00:01 GMT<div>R. Kandimalla</div><div>A.A. van Tilborg</div><div>E.C. Zwarthoff</div>
Urinary bladder cancer is the fifth most common cancer in the Western world. Increasing evidence has shown that DNA methylation in bladder cancer is expansive and is implicated in pathogenesis. Furthermore, distinct methylation patterns have been identified between non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC), as well as between FGFR3-mutant and wild-type tumours. Given these distinctions in expression, methylated genes have been proposed as diagnostic and prognostic biomarkers for patients with bladder cancer. Indeed, several studies have revealed that methylated genes-including CDH1, FHIT, LAMC2, RASSF1A, TIMP3, SFRP1, SOX9, PMF1 and RUNX3-are associated with poor survival in patients with MIBC. Further validation of these markers for prognostication as well as surveillance (of patients with NMIBC) is required. Validated markers for progression, diagnosis, survival and BCG response will contribute to clinical decision-making and individualized treatment.FGFR3 Mutation Analysis in Voided Urine Samples to Decrease Cystoscopies and Cost in Nonmuscle Invasive Bladder Cancer Surveillancehttp://repub.eur.nl/pub/39382/
Fri, 22 Mar 2013 00:00:01 GMT<div>K.E.M. van Kessel</div><div>L.C. Kompier</div><div>E.W. de Bekker-Grob</div><div>T.C.M. Zuiverloon</div><div>Y. Vergouwe</div><div>E.C. Zwarthoff</div><div>E.W. Steyerberg</div>
Purpose: We determined whether FGFR3 mutation analysis of voided urine samples would be cost-effective to partly replace cystoscopy in the surveillance of patients treated for nonmuscle invasive urothelial carcinoma. Materials and Methods: In this decision analytical study we analyzed data on 70 Dutch patients with FGFR3 positive primary tumors and a median followup of 8.8 years. Surveillance strategies were compared in a Markov model. Modified surveillance consisted of FGFR3 mutation analysis of voided urine samples every 3 months, and cystoscopy at 3, 12 and 24 months. Standard surveillance was defined as cystoscopy every 3 months and minimal surveillance was defined as cystoscopy at 3, 12 and 24 months. Analysis was stratified for 3 risk profiles, including surveillance after 1) the primary tumor, 2) the first to third recurrence and 3) the fourth recurrence or more. Sensitivity analysis was performed to evaluate the impact of variations in cost, sensitivity and specificity. Results: The probability of no recurrence after 2 years of surveillance after a primary tumor was higher for modified surveillance than for standard and minimal surveillance, eg after primary tumors (95.7% vs 95.0% and 93.9%, respectively). The total cost of surveillance after the primary tumor was lower for minimal and modified surveillance (€2,254 and €2,558, respectively) than for standard surveillance (€5,861). Results were robust to changing inputs over plausible ranges and consistent for each of the 3 risk profiles. Conclusions: Surveillance in which cystoscopy is partly replaced by FGFR3 mutation analysis of urine seems a safe, effective and cost-effective surveillance strategy. Further validation in larger cohorts is required.Analysis of molecular intra-patient variation and delineation of a prognostic 12-gene signature in non-muscle invasive bladder cancer; Technology transfer from microarrays to PCRhttp://repub.eur.nl/pub/73420/
Tue, 09 Oct 2012 00:00:01 GMT<div>L. Dyrskjot</div><div>T. Reinert</div><div>A. Novoradovsky</div><div>T.C.M. Zuiverloon</div><div>W. Beukers</div><div>E.C. Zwarthoff</div><div>N. Malats</div><div>F.X. Real</div><div>U. Segersten</div><div>P.-U. Malmström</div><div>M. Knowles</div><div>C. Hurst</div><div>J. Sorge</div><div>M. Borre</div><div>T.F. Orntoft</div>
Background: Multiple clinical risk factors and genetic profiles have been demonstrated to predict progression of non-muscle invasive bladder cancer; however, no easily clinical applicable gene signature has been developed to predict disease progression independent of disease stage and grade. Methods: We measured the intra-patient variation of an 88-gene progression signature using 39 metachronous tumours from 17 patients. For delineation of the optimal quantitative reverse transcriptase PCR panel of markers, we used 115 tumour samples from patients in Denmark, Sweden, UK and Spain. Results: Analysis of intra-patient variation of the molecular markers showed 71% similar classification results. A final panel of 12 genes was selected, showing significant correlation with outcome. In multivariate Cox regression analysis, we found that the 12-gene signature was an independent prognostic factor (hazard ratio=7.4 (95% confidence interval: 3.4-15.9), P<0.001) when adjusting for stage, grade and treatment. Independent validation of the 12-gene panel and the determined cut-off values is needed and ongoing. Conclusion: Intra-patient marker variation in metachronous tumours is present. Therefore, to increase test sensitivity, it may be necessary to test several metachronous tumours from a patients disease course. A PCR-based 12-gene signature significantly predicts disease progression in patients with non-muscle invasive bladder cancer.Prognostic value of molecular markers, sub-stage and European Organisation for the Research and Treatment of Cancer risk scores in primary T1 bladder cancerhttp://repub.eur.nl/pub/62648/
Mon, 01 Oct 2012 00:00:01 GMT<div>B.W. van Rhijn</div><div>L. Liu</div><div>A.N. Vis</div><div>P.J. Boström</div><div>T.C.M. Zuiverloon</div><div>N.E. Fleshner</div><div>M.N.M. van der Aa</div><div>S. Alkhateeb</div><div>C.H. Bangma</div><div>M.A.S. Jewett</div><div>E.C. Zwarthoff</div><div>B. Bapat</div><div>Th.H. van der Kwast</div><div>A.R. Zlotta</div>
Study Type - Prognosis (case series) Level of Evidence 4 What's known on the subject? and What does the study add? The stakes are high when making treatment decisions in T1 bladder cancer (BC). Conservative management may lead to progression and possibly death from BC. Conversely, radical cystectomy could be over-treatment of non-progressive disease. The problem for clinicians is that reliable prognostic indices are lacking. We performed a head-to-head comparison of two substaging systems, European Organisation for the Research and Treatment of Cancer (EORTC) risk scores and four molecular markers in T1 carcinomas of the bladder treated conservatively with BCG. T1 sub-stage according to a new system (micro-invasive [T1m] and extensive-invasive [T1e]) was the most important clinical variable for predicting progression to carcinoma invading bladder muscle. The performance of the EORTC risk scores was disappointing for this T1 sub-group. Molecular markers were not significant in multivariable analysis for predicting progression. Future studies may lead to the incorporation of sub-stage (T1m/T1e) in the TNM classification system for urinary BC to guide clinical decision-making in T1 BC. OBJECTIVE To evaluate the prognostic significance of four molecular markers, sub-stage and European Organisation for the Research and Treatment of Cancer (EORTC) risk scores in primary T1 bladder cancer (BC) treated with adjuvant bacille Calmette-Guérin. PATIENTS AND METHODS The slides of 129 carcinomas of the bladder from two university hospitals were reviewed and the T1 diagnosis was confirmed. T1 sub-staging was done in two separate rounds, using a new system that identifies micro-invasive (T1m) and extensive-invasive (T1e) T1BC, and then according to invasion of the muscularis mucosae (T1a/T1b/T1c). The EORTC risk scores for recurrence and progression were calculated. Uni- and multivariable analyses for recurrence and progression were performed using clinicopathological variables, T1 sub-stage, EORTC risk scores and molecular markers (fibroblast growth factor receptor 3 gene mutation and Ki-67, P53, P27 expression). RESULTS The median follow-up was 6.5 years. Forty-two patients remained recurrence-free (33%). Progression to T2 or metastasis was observed in 38 (30%) patients. In multivariable analysis for recurrence, multiplicity was significant. In multivariable analysis for progression, female gender, sub-stage (T1m/T1e) and carcinoma in situ (CIS) were significant. Molecular markers were significant in univariable and in multivariable analyses for recurrence. EORTC risk scores were not significant. CONCLUSIONS CIS, female gender and sub-stage (T1m/T1e) were the most important variables for progression. The additional value of molecular markers was modest. Sub-stage (T1m/T1e) could potentially be incorporated in future tumour-node-metastasis classifications.The Leukemia-Associated Fusion Protein MN1-TEL Blocks TEL-Specific Recognition Sequenceshttp://repub.eur.nl/pub/66882/
Wed, 26 Sep 2012 00:00:01 GMT<div>W.M. ter Haar</div><div>M.A. Meester-Smoor</div><div>K.H. van Wely</div><div>C.C.M.M. Schot</div><div>M.J.F.W. Janssen</div><div>B. Geverts</div><div>J. Bonten</div><div>G.C. Grosveld</div><div>A.B. Houtsmuller</div><div>E.C. Zwarthoff</div>
The leukemia-associated fusion protein MN1-TEL combines the transcription-activating domains of MN1 with the DNA-binding domain of the transcriptional repressor TEL. Quantitative photobleaching experiments revealed that ~20% of GFP-tagged MN1 and TEL is transiently immobilised, likely due to indirect or direct DNA binding, since transcription inhibition abolished immobilisation. Interestingly, ~50% of the MN1-TEL fusion protein was immobile with much longer binding times than unfused MN1 and TEL. MN1-TEL immobilisation was not observed when the TEL DNA-binding domain was disrupted, suggesting that MN1-TEL stably occupies TEL recognition sequences, preventing binding of factors required for proper transcription regulation, which may contribute to leukemogenesis.Selection of microsatellite markers for bladder cancer diagnosis without the need for corresponding bloodhttp://repub.eur.nl/pub/73433/
Wed, 22 Aug 2012 00:00:01 GMT<div>A.A. van Tilborg</div><div>L.C. Kompier</div><div>I. Lurkin</div><div>L.J. Poort</div><div>S. el Bouazzaoui</div><div>K.A. van der Keur</div><div>T.C.M. Zuiverloon</div><div>L. Dyrskjot</div><div>T.F. Orntoft</div><div>M.J. Roobol-Bouts</div><div>E.C. Zwarthoff</div>
Microsatellite markers are used for loss-of-heterozygosity, allelic imbalance and clonality analyses in cancers. Usually, tumor DNA is compared to corresponding normal DNA. However, normal DNA is not always available and can display aberrant allele ratios due to copy number variations in the genome. Moreover, stutter peaks may complicate the analysis. To use microsatellite markers for diagnosis of recurrent bladder cancer, we aimed to select markers without stutter peaks and a constant ratio between alleles, thereby avoiding the need for a control DNA sample. We investigated 49 microsatellite markers with tri- and tetranucleotide repeats in regions commonly lost in bladder cancer. Based on analysis of 50 blood DNAs the 12 best performing markers were selected with few stutter peaks and a constant ratio between peaks heights. Per marker upper and lower cut off values for allele ratios were determined. LOH of the markers was observed in 59/104 tumor DNAs. We then determined the sensitivity of the marker panel for detection of recurrent bladder cancer by assaying 102 urine samples of these patients. Sensitivity was 63% when patients were stratified for LOH in their primary tumors. We demonstrate that up-front selection of microsatellite markers obliterates the need for a corresponding blood sample. For diagnosis of bladder cancer recurrences in urine this significantly reduces costs. Moreover, this approach facilitates retrospective analysis of archival tumor samples for allelic imbalance.Down-staging (<pT2) of urothelial cancer at cystectomy after the diagnosis of detrusor muscle invasion (pT2) at diagnostic transurethral resection (TUR): Is prediction possible?http://repub.eur.nl/pub/69515/
Wed, 01 Aug 2012 00:00:01 GMT<div>W. Beukers</div><div>T. Meijer</div><div>K.J. Vissers</div><div>J.L. Boormans</div><div>E.C. Zwarthoff</div><div>G.J.H.L. Leenders</div>
Urothelial cell carcinoma (UCC) with musculus detrusor (MD) invasion is treated by cystectomy. Subsequent pathologic evaluation of cystectomies does not reveal MD invasion (<pT2) in a subgroup of patients. Our objective was to identify features at diagnostic transurethral resection (TUR) predicting down-staging (<pT2) at cystectomy. Patients with pathologically confirmed MD invasion at TUR followed by cystectomy for UCC without (neo-) adjuvant therapy were included (N=106). Slides of both TUR and cystectomy specimens were reviewed, and survival analyses were performed. In total, 27/106 (26 %) tumors were down-staged at cystectomy, of which 13 (12 %) had no residual tumor (pT0). There was no significant difference in age, gender, time interval between TUR and operation, number of slides sampled, and presence of TUR scar between down-staged (<pT2) and pT2 UCC. At review of TUR specimens (N=52) with UCC initially diagnosed as pT2, MD invasion was not confirmed in eight cases (15 %). One case showed extensive histiocytic reaction misinterpreted as UCC; in four cases, muscularis mucosae had been considered MD, and in three cases, desmoplastic reaction mimicked MD. No histologic parameter at TUR was significantly associated with down-staging at cystectomy. Overall and disease-specific survival was not statistically different in down-staged and pT2 UCC. In conclusion, down-staging of UCC (<pT2) at cystectomy occurred in 26 %. At review of diagnostic TURs, MD invasion was not confirmed in 15 %. No clinical or pathologic parameter was predictive for down-staging at cystectomy. There was no difference in survival between down-staged and pT2-staged UCC.Genome-wide analysis of CpG Island methylation in bladder cancer identified TBX2, TBX3, GATA2, and ZIC4 as pTa-specific prognostic markershttp://repub.eur.nl/pub/67936/
Fri, 01 Jun 2012 00:00:01 GMT<div>R. Kandimalla</div><div>A.A. van Tilborg</div><div>L.C. Kompier</div><div>D.J.P.M. Stumpel</div><div>R.W. Stam</div><div>C.H. Bangma</div><div>E.C. Zwarthoff</div>
Background: DNA methylation markers could serve as useful biomarkers, both as markers for progression and for urine-based diagnostic assays. Objective: Identify bladder cancer (BCa)-specific methylated DNA sequences for predicting pTa-specific progression and detecting BCa in voided urine. Design, setting, and participants: Genome-wide methylation analysis was performed on 44 bladder tumours using the Agilent 244K Human CpG Island Microarray (Agilent Technologies, Santa Clara, CA, USA). Validation was done using a custom Illumina 384-plex assay (Illumina, San Diego, CA, USA) in a retrospective group of 77 independent tumours. Markers for progression were identified in pTa (n = 24) tumours and validated retrospectively in an independent series of 41 pTa tumours by the SNaPshot method (Applied Biosystems, Foster City, CA, USA). Measurements: The percentage of methylation in tumour and urine samples was used to identify markers for detection and related to the end point of progression to muscle-invasive disease with Kaplan-Meier models and multivariate analysis. Results and limitations: In the validation set, methylation of the T-box 2 (TBX2), T-box 3 (TBX3), GATA binding protein 2 (GATA2), and Zic family member 4 (ZIC4) genes was associated with progression to muscle-invasive disease in pTa tumours (p = 0.003). Methylation of TBX2 alone showed a sensitivity of 100%, a specificity of 80%, a positive predictive value of 78%, and a negative predictive value of 100%, with an area under the curve of 0.96 (p < 0.0001) for predicting progression. Multivariate analysis showed that methylation of TBX3 and GATA2 are independent predictors of progression when compared to clinicopathologic variables (p = 0.04 and p = 0.03, respectively). The predictive accuracy improved by 23% by adding methylation of TBX2, TBX3, and GATA2 to the European Organisation for Research and Treatment of Cancer risk scores. We further identified and validated 110 CpG islands (CGIs) that are differentially methylated between tumour cells and control urine. The limitation of this study is the small number of patients analysed for testing and validating the prognostic markers. Conclusions: We have identified four methylation markers that predict progression in pTa tumours, thereby allowing stratification of patients for personalised follow-up. In addition, we identified CGIs that will enable detection of bladder tumours in voided urine.A methylation assay for the detection of non-muscle-invasive bladder cancer (NMIBC) recurrences in voided urinehttp://repub.eur.nl/pub/69471/
Thu, 01 Mar 2012 00:00:01 GMT<div>T.C.M. Zuiverloon</div><div>W. Beukers</div><div>K.A. van der Keur</div><div>J.R. Munoz</div><div>C.H. Bangma</div><div>H.F. Lingsma</div><div>M.J.C. Eijkemans</div><div>J.P. Schouten</div><div>E.C. Zwarthoff</div>
OBJECTIVE To develop a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay for the detection of non-muscle invasive bladder cancer (NMIBC) recurrences in voided urine. PATIENTS AND METHODS Genes frequently methylated in NMIBC tumours (n= 37) were selected to develop a BC-specific MS-MLPA assay. Genes methylated in blood from patientswith BC (n= 29) and genes methylated in urine from patients with no history of BC (n= 46) were excluded. A four-gene panel with the highest predictive value was selected from the initial assay. This four-gene panel was tested and validated on urine from patients with a histologically confirmed recurrence (n= 68 test set; n= 49 validation set) and urine samples from patients without BC (n= 91, test set) and urine from recurrence-free BC (rec-free BC) patients (n= 60, validation set). A model was developed to predict the probability of having a recurrence based on methylation of the four-gene panel and a threshold probability with the highest sensitivity and specificity was determined. The outcome of the model was validated on BC urine samples (n= 65) and on urine samples from rec-free BC patients (n= 29). RESULTS The BC MS-MLPA assay consisted of 23 methylation probes. The selected four-gene panel included: APC-a, TERT-a, TERT-b, and EDNRB. This panel reached an area under the receiver operating characteristic curve (AUC) of 0.82 (test set) and AUC 0.69 (validation set). Sensitivity and specificity for the detection of a concomitant tumour were 63.3% and 58.3% respectively (test set) and 72.3% and 55.2%, respectively (validation set). CONCLUSIONS We have developed a methylation detection assay specifically for the detection of recurrences in patients with NMIBC in voided urine. The findings are promising and improvement of this test could eventually contribute to a more individualized patient friendly surveillance.